GSM6726931: Day 3, post-injury, scRNAseq; Mus musculus; RNA-Seq
In brief, carotids were cut in small pieces and digested for 1h at 37°C with an enzyme mix containing collagenase type XI (125 U/ml), collagenase type I (450 U/ml), hyaluronidase (60 U/ml) and deoxyribonuclease I (60 U/ml). Digestion was stopped with stopping solution (PBS; 2% FCS; 1mM EDTA) and cell suspension was strained. Dead cells were removed by MACS Dead cell removal kit (Miltenyi, Germany). Cells were washed and resuspended in PBS and used for droplet scRNA-seq. Cellular suspensions were loaded on a 10x Chromium Controller (10x Genomics) according to manufacturer's protocol. All scRNA-seq libraries were prepared using Chromium Single Cell 30 v2 Reagent Kit (10x Genomics) according to manufacturer's protocol. In brief, the initial step consisted in performing an emulsion where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10x cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Germany). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98°C for 3min; cycled 14x: 98°C for 15 s, 67°C for 20 s, and 72°C for 1min; 72°C for 1min; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter, USA). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 30 v2 Reagent Kit, as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads. Library quantification and quality assessment were performed using Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (Agilent Genomics, USA).